Differences exist within the literature as to what constitutes the important variables in the design of hybrid toxins with altered receptor specificity. As originally conceived, the binding chain (B chain) of diphtheria toxin or ricin was replaced with an alternate binding protein modified to form a disulfide linkage with the toxin A chain. The theoretical advantage of this scheme was that toxin A chains have undetectable binding to cell membranes, and therefore the specificity of the hybrid would be directed entirely by the new binding moiety. Initial results with both diphtheria toxin and ricin A chain hybrids of peptide hormones were disappointing; under conditions which tested selectivity between target and non-target cells, potency of the hybrid relative to the parent toxin was markedly reduced. Certain antibody-toxin A chain hybrids showed good selectivity but were still less toxic than the parent toxin.
Subsequent discoveries showed the feasibility of altering the receptor specificity of intact toxins such as ricin by attaching a new binding moiety and inhibiting the usual ricin entry route by competition with lactose; U.S. Ser. No. 186,735 filed Sept. 12, 1980; U.S. Ser. No. 199,781 filed Oct. 23, 1980; and U.S. Ser. No. 341,572 filed Jan. 21, 1982. These hybrids in the presence of lactose were as toxic as ricin in the absence of lactose when assayed after a three-hour exposure to cells. These hybrids required a functional galactose binding site on the ricin B chain for optimal specific toxicity. Recently, however, several A chain hybrids have been reported which show a high degree of selectivity and are as potent as the parent toxins when assayed after 18-26 hours in tissue culture.
The present invention shows the rates of inactivation of protein synthesis between 2 and 24 hours for two A chain hybrids with widely differing affinities for the alternate receptor. The alternate binding proteins are monoclonal antibodies which bind the Thy 1.1 antigen present on AKR cells. Ricin B chain properties are studied by adding the B chain to mixtures of the A chain hybrids and target cells.
The protein inhibition rate is much lower for Thy 1.1 ricin A chain hybrid than for ricin alone. Additionally, the rate of inhibition by the hybrid can be increased by adding excess ricin B chain with no change in the amount of A chain bound to the target cells. In other words, ricin B chain facilitates entry of the A chain into the cytosol compartment of the cell by a process independent of the amount of A chain bound to the surface membrane.